This project is focused on the two of the most important issues in RNA editing in kinetoplastids; determining the mechanism by which the edited sequence is specified and definitively identifying and characterizing the macromolecular machinery that catalyzes editing (the editosome). It will use the in vitro editing system that was developed in the previous grant period and develop transgenic trypanosomes to characterize the critical catalytic steps in the editing reaction series in order to determine which steps specify the number of uridylates that are inserted and deleted. It will utilize the monoclonal antibodies that specifically immunopurify in vitro editing and its associated catalytic activities, the cloned gBP21 gene, and biochemical purification procedures developed during the previous grant period to identify additional editosome components, clone their genes and use these to study their function in editing and editosome assembly. Aim 1 will identify nucleotieds in gRNA and mRNA that are required for accurate and efficient RNA editing. It will develop minimal gRNA and pre-mRNA substrates, alternative RNA substrates including those that mimic the last editing site, and RNA substrates that support partial editing reactions. Editing of novel cis and "precleaved" RNA substrates will be assessed since they will be valuable for analysis of specific editing steps. Aim 2 will independently examine critical catalytic steps in editing to determine if the edited sequence is specified at the endoribonuclease, uridylate addition/deletion, or RNA ligation steps, or some combination thereof. It will use conditions that block or pause editing at specific catalytic steps and mutations in the substrate RNAs to determine the role of each catalytic activity in specifying accurate U insertion and deletion. Aim 3 is directed at identifying additional editosome components, cloning their genes and assessing their function by gene KO and inactivation. The genes will identified by biochemical fractionation, crosslinking, monoclonal antibody development, and yeast two and three hybrid approaches. The potential editosome components will be assessed by a variety of methods and priority candidates will be selected for gene knockout and gene inactivation experiments that will assess the specific functions of the genes. Aim 4 is focused on determining the general structure of the editosome, including whether it is composed of discrete subunits, and exploring assembly of the functional editosome. It will utilize the tools and materials developed in the earlier aims including the ability to block or stall editing at specific steps. This project is designed to determine the critical features of a novel and fundamental genetic regulatory process that occurs at the RNA level. The resultant knowledge should expand our understanding of the diversity of basic molecular genetic and regulatory processes which may occur in a variety of organisms.